Defects in mitochondrial biogenesis lead to a broad range of diseases including neurodegeneration, stroke, myocardial infarction, ischemia, and cancer;however, therapies to correct such diseases are not readily available. We propose to conduct a high throughput screen in the Molecular Libraries Production Center Network (MLPCN) identical to the one that, on a smaller screening scale, has already successfully identified inhibitors of the mitochondrial TIM22 protein import pathway that is required for the assembly of inner membrane proteins in the model organism Saccharomyces cerevisiae. Specifically, we have devised a growth-based assay using a yeast temperature-sensitive mutant that is compromised for growth at a high temperature because of a defective import system. In the screen, we select for small molecules that are synthetically lethal with the mutant strain at a temperature that normally permits growth. Using this assay, we have identified small molecules that specifically target the TIM22 import pathway in yeast mitochondria. The aims of this proposal are to (1) identify small molecules that target the TIM22 import pathway and alter its function and then develop analogs for structure activity relationship (SAR) studies to identify specific chemical compounds that modulate this pathway and (2) utilize these tools in secondary assays and develop probes that we can translate to vertebrate systems to probe mitochondrial function, including the link to mitochondrial diseases, because protein import is highly conserved from yeast to mammals. These studies will result in validated chemical probes for mechanistic studies of mitochondrial import and for potentially inducing/abrogating mitochondrial diseases. A defective TIM22 import pathway leads to the inherited disease, deafness-dystonia syndrome, which results in neurodegeneration. Given our success, we are confident that many novel compounds will be identified that are pertinent for understanding mitochondrial assembly in vertebrates and potentially serving as tools to characterize the molecular basis of deafness-dystonia syndrome. Generally, the medical importance of events regulated by mitochondrial assembly, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for diseases affected by dysfunctional mitochondria. Identification of these novel compounds, tied with our expertise in finding targets and our ability to exploit them to more fully understand mechanism, justifies our request to expand this screen through the MLPCN. This study is relevant to public health because it may lead to the development of new therapeutics for degenerative muscular and neural diseases. PUBLIC HEALTH RELEVANCE: This project will develop small molecules as probes to investigate the cause of neurodegenerative and degenerative muscular diseases that are initiated by defects in mitochondrial function, using yeast as a model system because protein import pathways are highly conserved from yeast to mammals. The mitochondrion generates energy for the cell and is linked to a broad range of diseases, including cancer and degenerative muscular and neural diseases. Long-term, this project may lead to the development of therapeutics that modulates mitochondrial function in these diseases.